Thymic-derived T regulatory cells (Treg) express the lineage commitment transcription factor Foxp3 and play critical roles in all aspects of immune responses including control of the interaction of the host with microbes. The mechanisms by which Tregs modulate responses to infectious agents are complex and include limiting exuberant inflammatory responses with control of tissue damage, facilitation of the persistence of the pathogen for maintenance of immunity, but also enhanced pathogen replication and suppression of effector T cell function. Progress in this area has been limited because of our lack of knowledge regarding the nature of the antigens recognized by Tregs. The mouse model of infection with lymphocytic choriomeningitis virus (LCMV) allows one to differentiate between the effects of acute and chronic viral infection on Treg activation. Our initial approach to exploring the role of Treg during LCMV infection was to compare the frequency and phenotype of Treg in C57BL/6 mice during the course of Armstrong and Clone 13 LCMV infection. Treg dramatically increased in frequency among splenic CD4+CD8- T cells during Clone 13, but not Armstrong, infection. This increase in the frequency of Foxp3+ Treg was transient and peaked at 17 days post infection (dpi). Treg from Clone 13 infected mice displayed a much more activated phenotype. A marked increase in the frequency of Foxp3+ Treg expressing TCR Vbeta5 was observed, again specifically during Clone 13 infection. There was no change in the percentage of Treg expressing any of the other Vbeta analyzed and no change in the frequency of CD4+Vbeta5+Foxp3- cells was noted during either Armstrong or Clone 13 infections. The failure to observe Treg expansion during Armstrong infection was secondary to the absence of viral chronicity and not to the minor sequence difference between the two strains. Studies in other infectious disease models suggest that Tregs in infected animals may be specific for the infectious organism or perhaps specific for ubiquitous or tissue-specific antigens whose expression is enhanced during the course of the infection. We saw no evidence for Treg cell specific reactivity in response to a library of overlapping peptides that span the entire LCMV proteome. Because the expansion of the Treg in mice chronically infected with LCMV was restricted to a specific Vbeta segment, we also considered the possibility that a superantigen (Sag) may be driving proliferation. As LCMV has not been previously shown to express a superantigen, a likely explanation for our results was that the expansion of the Vbeta5+ Treg was secondary to stimulation by an endogenous Sag such as mouse mammary tumor virus-(Mtv) encoded Sags. To examine this possibility, we infected BALB/c, that normally express Mtv-6, -8, and -9 and delete Vbeta-5, -11, and -12 T cells in the thymus, with Clone 13 and analyzed the effects of infection on the Vbeta repertoire. We observed a significant increase in the percentage of CD4+Foxp3+Vbeta5+ and a dramatic increase in Foxp3+Vbeta12+ Treg, but no change in the percentage of CD4+Foxp3-Vbeta5+ or Foxp3-Vbeta12+ T cells, following Clone 13 infection. To directly prove that expansion of the Vbeta5 and Vbeta12 Foxp3+ subsets in BALB/c mice was secondary to activation by endogenous Mtv-Sag, we infected BALB/c mice lacking all three of the endogenous Mtv proviruses (Mtv-null mice). Consistent with a lack of Mtv-Sag, these mice had a substantial population of T cells that expressed either Vbeta5 or Vbeta12 in Foxp3+ and Foxp3- subsets. However, we observed no change in the frequencies of either Vbeta5+ or Vbeta12+ Treg in these mice following infection, thus demonstrating that the observed Vbeta-specific Treg expansion was, in fact, due to stimulation by an endogenous retroviral superantigen. We have focused on determining the mechanism by which chronic infection results in Mtv Sag-dependent Treg expansion. In particular, we have sought to identify the antigen presenting cell (APC) responsible for Sag-dependent Treg expansion. First, we asked whether LCMV infection resulted in increased expression of the endogenous Sag genes. Because Mtv8 and Mtv9 Sag appeared to be responsible for the Vbeta-specific Treg expansion, we determined the relative expression of these genes in splenic MHC class II-enriched cells from BALB/c mice by quantitative real time-PCR. At 8 days post infection, Mtv8 and/or Mtv9 Sag expression was up regulated 2- and 25-fold in Armstrong and clone 13 infected mice, respectively, compared to uninfected controls. To further define the type of APC responsible for Treg expansion, we examined the role of B cells and dendritic cells (cell types that have been shown to be the primary APC that express several endogenous Mtvs in the periphery) using genetically deficient mice. Infection of B cell deficient mice (&#956;MT) resulted in normal expansion similar to wild-type mice. However, Flt3L-/- mice (which are defective in several hematopoietic cell lineages, but primarily dendritic cells) infected with clone 13 displayed only a modest increase in frequency of Vbeta-specific Treg, suggesting that Sag presentation by dendritic cells was primarily responsible for the Treg expansion observed following infection. Because dendritic cells have the potential to produce a plethora of pro-inflammatory cytokines, we further hypothesized that mice unable to make individual pro-inflammatory cytokines may display a defect in Treg expansion following infection. To test this, we infected IL-1Ralpha-/-, IL-6-/-, IL-12 (p35)-/-, TNFalpha-/-, or IFNalpha-betaR-/- mice with clone 13 and analyzed the effect on Vbeta-specific Treg expansion. Only IFNalpa-betaR-/- mice infected with clone 13 showed a lack of Vbeta-specific Treg expansion following infection. Stimulation of dendritic cells through toll-like receptors (TLR) is a primary mechanism by which Type-I IFNs are produced. This mechanism also appeared to be important in LCMV-mediated Treg expansion as infection of MyD88-/- mice with clone 13 resulted in a similar lack of expansion of Vbeta5-specific Treg, suggesting that MyD88-dependent Type-I IFN production is required for Mtv Sag-dependent Treg expansion following clone 13 infection.